Use of mulberry extract in preparation of medicament for treating and / or preventing diabetic vascular complications

Abstract

According to the application of the mulberry extract in preparing the medicine for treating and / or preventing the diabetic vascular complications, the mulberry extract comprises alkaloid with the weight content being 3% or above, and / or polysaccharide with the weight content being not higher than 70%, flavone with the weight content being not higher than 10%, and / or amino acid with the weight content being not higher than 50%. And / or other components. The mulberry extract disclosed by the invention has a remarkable improvement effect on vascular complications of diabetes mellitus.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202410675192.X (22) Application Date 2024.05.28 (71) Applicant Beijing Wuhe Boao Pharmaceutical Co., Ltd. Address 30 Tianfu Street, Daxing Biomedical Industry Base, Zhongguancun Science Park, Daxing District, Beijing 102600 Applicant Guangxi Wuhe Boao Pharmaceutical Co., Ltd. (72) Inventors Liu Yuling, Deng Wuquan, Wang Tingting, Rui Shunli, Xiao Fugang, Zhu Xiangyang, Liu Zhihua, Chen Qiu, Jiang Xiaoyan, Chen Yanmin, Chen Yan, Deng Bo, Liu Han (74) Patent Agency Beijing Tanlulai Patent Agency Co., Ltd. 11652 Patent Attorney Sheng Shujuan (51) Int.Cl. A61K 36 / 605 (2006.01) A61P 9 / 00 (2006.01) A61P 3 / 10 (2006.01) A61P 9 / 14 (2006.01) A61P 17 / 02 (2006.01) A61P 9 / 10 (2006.01) (54) Invention Title: Use of Mulberry Extract in the Preparation of a Drug for Treating and / or Preventing Diabetic Vascular Complications (57) Abstract: Use of mulberry extract in the preparation of a drug for treating and / or preventing diabetic vascular complications, wherein the mulberry extract contains alkaloids at a weight content of more than 3%, and / or contains polysaccharides at a weight content of not more than 70%, and / or contains flavonoids at a weight content of not more than 10%, and / or contains amino acids at a weight content of not more than 50%, and / or other components. The mulberry extract of the present invention has a significant effect on improving diabetic vascular complications. Claims (5 pages), Description (23 pages), Drawings (11 pages), CN 121015729 A, 2025.11.28, CN 1 21 01 57 29 A. 1. Use of mulberry extract in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that, based on the weight percentage of the sum of all components of the mulberry extract as 100%, the mulberry extract contains at least 3% alkaloids by weight (optionally containing 3-99% alkaloids by weight, further optionally containing 15-99% alkaloids by weight, further optionally containing 30-99% alkaloids by weight, further optionally containing 40-99% alkaloids by weight, further optionally containing 50-99% alkaloids by weight, further optionally containing 60-99% alkaloids by weight). And / or contains polysaccharides with a weight content not exceeding 70% (optionally containing 0.2-70% polysaccharides by weight, further optionally containing 0.2-50% polysaccharides by weight, further optionally containing 0.2-35% polysaccharides by weight, and so on).The step may optionally include 0.2-25% polysaccharides by weight, further optionally 0.2-23% polysaccharides by weight, further optionally 20-25% polysaccharides by weight, and / or include no more than 10% flavonoids by weight (optionally 0.05-5% flavonoids by weight, further optionally 0-2% flavonoids by weight, further optionally 0.05-2% flavonoids by weight, further optionally 0.5-1.5% flavonoids by weight, further optionally 0-1% flavonoids by weight, further optionally 0.05-1% flavonoids by weight). And / or contains amino acids at a weight content not exceeding 50% (optionally containing 0-30% amino acids, further optionally containing 0-25% amino acids, further optionally containing 0-20% amino acids, further optionally containing 0-5% amino acids, further optionally containing 3-25% amino acids, or further optionally containing 5-20% amino acids), and / or other components (optionally 0-25% by weight, further optionally 0-20% by weight, further optionally 0-15% by weight, further optionally 0-11% by weight, further optionally 2-20% by weight, further optionally 4-8% by weight). 2. The use of the mulberry extract according to claim 1 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that the treatment and / or prevention of diabetic vascular complications is manifested in improving vascular endothelial cell dysfunction; preferably specifically manifested in improving any one or more of the following: (1) promoting vascular endothelial cell proliferation; (2) promoting vascular endothelial cell migration; (3) promoting angiogenesis; (4) inhibiting vascular endothelial cell apoptosis; (5) promoting NO accumulation in endothelial cells; (6) upregulating the expression of vasoactive factors, preferably the vasoactive factors include CD31 (platelet-endothelial cell adhesion factor-1), VEGF (vascular endothelial growth factor) (preferably VEGF-A) and / or eNOS (endothelial nitric oxide synthase); (7) alleviating vascular endothelial inflammation, preferably by inhibiting the adhesion of vascular endothelial cells to monocytes, preferably by inhibiting the secretion of vascular endothelial cell adhesion factors, the adhesion factors including P-selectin and / or E-selectin; (8) Increases the number of mitochondria in vascular endothelial cells. 3. The use of the mulberry extract according to claim 2 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that the improvement of vascular endothelial cell function is further manifested by promoting the expression of NRF2 (nuclear factor erythroid lineage 2-related factor 2) and / or HO-1 (heme oxygenase 1). ClaimsPage 1 / 5 2 CN 121015729 A 4. Use of the mulberry extract according to claim 1 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that the diabetic vascular complications include diabetic foot or diabetic atherosclerosis. 5. Use of the mulberry extract according to claim 4 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that the treatment and / or prevention of diabetic foot is manifested by any one or more of the following: (1) promoting wound healing, including promoting wound regeneration, re-epithelialization and / or collagen production, (2) reducing cell apoptosis in the wound area, (3) promoting cell migration and / or proliferation in the wound area, (4) promoting angiogenesis, remodeling and / or repair in the wound area, preferably promoting the expression of CD31, VEGF (preferably VEGF-A) and / or eNOS. 6. The use of the mulberry extract according to claim 4 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that the treatment and / or prevention of diabetic atherosclerosis is manifested in any one or more of the following: (1) reducing arterial plaque formation, (2) improving arterial blood flow, (3) alleviating vascular endothelial inflammation, preferably, reducing the level of vascular endothelial cell adhesion factors, said adhesion factors including P-selectin and / or E-selectin. 7. The use of the mulberry extract according to any one of claims 1-6 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that, based on the weight percentage of the sum of the components of the mulberry extract as 100%, the weight content of each component in the mulberry extract is: Preferably, based on the weight percentage of the sum of the components of the mulberry extract as 100%, the weight content of each component in the mulberry extract is: Preferably, based on the weight percentage of the sum of the components of the mulberry extract as 100%, the weight content of each component in the mulberry extract is: Preferably, based on the weight percentage of the sum of the components of the mulberry extract as 100%, the weight content of each component in the mulberry extract is: Preferably, based on the weight percentage of the sum of the components of the mulberry extract as 100%, the weight content of each component in the mulberry extract is: Preferably, based on the weight percentage of the sum of the components of the mulberry extract as 100%, the weight content of each component in the mulberry extract is: Preferably, based on the weight percentage of the sum of the components of the mulberry extract as 100%, the weight content of each component in the mulberry extract is: Claims 3 / 5, page 4, CN 121015729 A. Preferably, based on the weight percentage of the sum of all components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is: More preferably, based on the weight percentage of the sum of all components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is:More preferably, based on the weight percentage of the sum of all components of the mulberry extract as 100%, the weight content of each component in the mulberry extract is as follows: Claims 4 / 5, page 5, CN 121015729 A 8. Use of the mulberry extract according to any one of claims 1-7 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that the alkaloids comprise 1-deoxynojirimycin (DNJ), N-methyl-1-deoxynojirimycin (N-methly-1-deoxynojirimycin), fagomine (FAG), 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), calystegin B2, and calystegin C1. One or more of 2-O-(α-D-galactopyranosyl)-1-deoxynojirimycin, 6-O-(β-D-glucopyranosyl)-1-deoxynojirimycin, and 1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol; optionally, the weight percentage of DNJ is not less than 50% (optionally 60-99%) of the total alkaloids. 9. Use of the mulberry extract according to any one of claims 1-8 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that the preparation of the mulberry extract comprises the following steps: 1) preparing a crude extract of a mulberry plant; 2) separating the crude extract with a cation exchange resin and / or optionally an anion exchange resin to obtain a resin eluent, optionally step 3) subjecting the resin eluent of step 2) to alcohol precipitation and collecting the supernatant; 4) concentrating and / or drying the supernatant; optionally, concentration and / or drying are performed before alcohol precipitation of the resin eluent of step 2). 10. Use of the mulberry extract according to any one of claims 1-9 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that the mulberry extract acts on humans or mammals; optionally, the medicament is an oral dosage form; optionally, the medicament is a tablet, capsule, oral solution, or oral administration.Emulsions, pills, granules, syrups, or powders. Claims 5 / 5 pages 6 CN 121015729 A Use of mulberry extract in the preparation of medicaments for the treatment and / or prevention of diabetic vascular complications Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to the use of mulberry extract in the preparation of medicaments for the treatment and / or prevention of diabetic vascular complications. Background Art

[0002] The International Diabetes Federation reported in the 10th edition of the Global Diabetes Atlas released at the end of 2021 that diabetes is currently one of the fastest growing global public health emergencies. Diabetes is a chronic metabolic disease; hyperglycemia does not cause death, but its complications can lead to a risk of death.

[0003] The pathogenesis of diabetic vascular complications is a complex process involving multiple factors, including hyperglycemia, advanced glycation end products (AGEs), oxidative stress, and inflammation. Long-term hyperglycemia can damage vascular endothelial cells, leading to vascular dysfunction and increased permeability. At the same time, hyperglycemia can lead to the accumulation of AGEs formed by the combination of glucose and protein in the blood vessel walls. In addition, diabetic patients often experience increased oxidative stress, leading to increased free radical production and damage to vascular endothelial cells and vessel walls. In general, the various pathophysiological changes caused by hyperglycemia are the main mechanisms for the occurrence of diabetic vascular complications, and vascular damage and abnormal blood circulation play important roles in this process.

[0004] Endothelial cells are a continuous monolayer of cells covering the inner walls of arteries and veins at all levels. Their total area is 400 square meters. In addition to their mechanical barrier function, vascular endothelial cells can also synthesize and secrete substances that regulate the coagulation-fibrinolysis system, factors that regulate vascular tone, and cytokines, playing an important role in angiogenesis, wound healing, antithrombosis, and inflammatory responses. Therefore, once endothelial function is impaired, it will affect various organs and multiple functions. Hyperglycemia is an important factor affecting vascular endothelial function, and at the same time, vascular endothelial cell dysfunction is the initiating factor of diabetic vascular complications. Both contribute to the occurrence and development of the disease. Addressing vascular endothelial damage caused by hyperglycemia has become an important approach to the prevention and treatment of diabetic vascular complications.

[0005] Experimental studies and clinical applications have confirmed that intensive blood glucose control does not reduce the incidence of diabetic vascular complications, and treatment with hypoglycemic drugs alone cannot achieve satisfactory therapeutic effects; some complications may still develop. The application of drugs with multiple vascular endothelial protective effects is an effective treatment strategy, achieving the effect of treating diabetic vascular complications on the basis of improving vascular endothelial cell function. Summary of the Invention

[0006] The present invention aims to provide the use of mulberry extract in the preparation of drugs for treating and / or preventing diabetic vascular complications.

[0007] In one embodiment of the present invention, a method for treating and / or preventing diabetic vascular complications is provided, comprising administering an effective amount of mulberry extract to the desired subject.

[0008] In one embodiment of the present invention, the use of a mulberry extract in the treatment and / or prevention of diabetic vascular complications is provided.

[0009] In one embodiment of the present invention, a mulberry extract is also provided for the treatment and / or prevention of diabetic vascular complications. Instruction manual, page 1 / 23, CN 121015729 A

[0010] In this invention, based on the weight percentage of the sum of the components of the mulberry extract as 100%, the mulberry extract contains alkaloids with a weight content of more than 3% (optionally containing alkaloids with a weight content of 3-99%, further optionally containing alkaloids with a weight content of 15-99%, further optionally containing alkaloids with a weight content of 30-99%, further optionally containing alkaloids with a weight content of 40-99%, further optionally containing alkaloids with a weight content of 50-99%, further optionally containing alkaloids with a weight content of 60-99%),

[0011] and / or contains polysaccharides with a weight content of not more than 70% (optionally containing polysaccharides with a weight content of 0.2-70%, further optionally containing polysaccharides with a weight content of 0.2-50%, further optionally containing polysaccharides with a weight content of 0.2-35%), further optionally containing polysaccharides with a weight content of 0. 0.2-25% polysaccharides, further optionally including 0.2-23% polysaccharides by weight, further optionally including 20-25% polysaccharides by weight),

[0012] and / or including no more than 10% flavonoids by weight (optionally including 0.05-5% flavonoids by weight, further optionally including 0-2% flavonoids by weight, further optionally including 0.05-2% flavonoids by weight, further optionally including 0.5-1.5% flavonoids by weight, further optionally including 0-1% flavonoids by weight, further optionally including 0.05-1% flavonoids by weight),

[0013] and / or including no more than 50% amino acids by weight (optionally including 0-30% amino acids by weight). Further optionally, it may contain 0-25% amino acids by weight, further optionally, it may contain 0-20% amino acids by weight, further optionally, it may contain 0-5% amino acids by weight, further optionally, it may contain 3-25% amino acids by weight, or further optionally, it may contain 5-20% amino acids by weight,

[0014] and / or other components (optionally 0-25% by weight, further optionally 0-20% by weight, further optionally 0-15% by weight, further optionally 0-11% by weight, further optionally 2-20% by weight, further optionally 4-8% by weight).

[0015] In a preferred embodiment of the invention, the treatment and / or prevention of diabetic vascular complications manifests asImprovement of vascular endothelial cell function; preferably specifically manifested as improvement of any one or more of the following:

[0016] (1) Promoting vascular endothelial cell proliferation;

[0017] (2) Promoting vascular endothelial cell migration;

[0018] (3) Promoting angiogenesis;

[0019] (4) Inhibiting vascular endothelial cell apoptosis;

[0020] (5) Promoting NO accumulation in endothelial cells;

[0021] (6) Upregulating the expression of vasoactive related factors, preferably the vasoactive related factors include CD31 (platelet-endothelial cell adhesion factor-1), VEGF (vascular endothelial growth factor) (preferably VEGF-A) and / or eNOS (endothelial nitric oxide synthase);

[0022] (7) Alleviating vascular endothelial inflammation, preferably by inhibiting the adhesion of vascular endothelial cells to monocytes, and more preferably by inhibiting the secretion of vascular endothelial cell adhesion factors, the adhesion factors including P-selectin and / or E-selectin;

[0023] (8) Increase the number of mitochondria in vascular endothelial cells.

[0024] Further, the treatment and / or prevention of diabetic vascular complications works by improving vascular endothelial cell dysfunction caused by high glucose, high insulin, and / or inflammatory conditions, wherein the high glucose conditions include high concentrations of glucose and / or glycosylation products (such as advanced glycation end products AGEs), and the inflammatory conditions include induction and / or stimulation by inflammatory factors (such as TNF-α).

[0025] Preferably, the improvement of vascular endothelial cell dysfunction is also manifested in promoting the expression of NRF2 (nuclear factor erythrocyte lineage 2) and / or HO-1 (heme oxygenase 1).

[0026] In a preferred embodiment of the present invention, the diabetic vascular complications include diabetic foot or diabetic atherosclerosis.

[0027] In a preferred embodiment, the diabetic foot includes no ulcer or ulceration, with accompanying infection or gangrene.

[0028] Preferably, the treatment and / or prevention of diabetic foot is manifested in any one or more of the following:

[0029] (1) promoting wound healing, including promoting wound regeneration, re-epithelialization and / or collagen production,

[0030] (2) reducing cell apoptosis in the wound area,

[0031] (3) promoting cell migration and / or proliferation in the wound area,

[0032] (4) promoting angiogenesis, remodeling and / or repair in the wound area, preferably promoting the expression of CD31, VEGF (preferably VEGF-A) and / or eNOS.

[0033] Preferably, the treatment and / or prevention of diabetic atherosclerosis is manifested in any one or more of the following:

[0034] (1) reducing arterial plaque formation,

[0035] (2) improving arterial blood flow,

[0036] (3) Relieve vascular endothelial inflammation, preferably by reducing the level of vascular endothelial cell adhesion factors, including P-selectin and / or E-selectin.

[0037] In a preferred embodiment of the present invention, with the total weight percentage of the components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows:

[0038]

[0039] Preferably, with the total weight percentage of the components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows:

[0040]

[0041] Preferably, with the total weight percentage of the components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows: Specification 3 / 23 Page 9 CN 121015729 A

[0042]

[0043] Preferably, with the total weight percentage of the components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows:

[0044]

[0045]

[0046] Preferably, based on the weight percentage of the sum of all components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows:

[0047]

[0048] Preferably, based on the weight percentage of the sum of all components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows:

[0049] Specification 4 / 23 pages 10 CN 121015729 A

[0050] Preferably, based on the weight percentage of the sum of all components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows:

[0051]

[0052]

[0053] More preferably, based on the weight percentage of the sum of all components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows:

[0054]

[0055] More preferably, based on the weight percentage of the sum of all components of the mulberry extract being 100%, the weight content of each component in the mulberry extract is as follows:

[0056]

[0057] Preferably, the alkaloid comprises 1-deoxynojirimycin (DNJ), N-methyl-1-deoxynojirimycin, fagomine (FAG), 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), calystegin B2, calystegin C1, 2-oxo-(α-D-)One or more of the following: galactopyranosyl)-1-deoxynojirimycin (2-O-(α-D-galactopyranosyl)-1-deoxynojirimycin), 6-oxo-(β-D-glucopyranosyl)-1-deoxynojirimycin (6-O-(β-D-glucopyranosyl)-1-deoxynojirimycin), and 1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol.

[0058] Preferably, the weight percentage of DNJ is not less than 50% (preferably 60-99%) of the alkaloid. Instructions for Use, Page 5 / 23, CN 121015729 A

[0059] Preferably, the heavy metal content of the mulberry extract does not exceed 10 ppm.

[0060] The drug is an oral dosage form; optionally, the drug is a tablet, capsule, tablet-containing tablet, powder, oral solution, oral emulsion, pill, granule, syrup or powder.

[0061] In this invention, the mulberry extract can be provided in the form of commercially available mulberry twig alkaloid tablets (National Drug Approval Number Z20200002).

[0062] Optionally, the mulberry extract can be prepared according to the method described in CN 110393738A.

[0063] Optionally, in this invention, the preparation of the mulberry extract includes the following steps:

[0064] 1) preparing a crude extract of mulberry plants; 2) separating the crude extract with a cation exchange resin and / or optionally an anion exchange resin to obtain a resin eluent; optional step 3) precipitating the resin eluent from step 2) with alcohol and collecting the supernatant; 4) concentrating and / or drying the supernatant. Optionally, before precipitating the resin eluent from step 2), concentration and / or drying are performed.

[0065] Preferably, the extraction method further includes: before the separation in step 2), concentrating the crude plant extract;

[0066] preferably, concentrating the crude plant extract to a solid content of 1-15%, preferably 2-10%. The solid content refers to the solid substance remaining after the water in the solution is removed.

[0067] Optionally, before the resin separation treatment in step 2), the concentrated crude extract can be subjected to alcohol precipitation. During alcohol precipitation, ethanol is added to the crude extract, stirred until well mixed, and then allowed to stand for a certain period of time to allow the insoluble substances to precipitate. Optionally, the volume-to-mass ratio of the added ethanol to the plant raw material is 0.2-20 times, preferably 0.4- (L / kg).10 times. Further, optionally, an alcohol precipitation tank is used for alcohol precipitation. Optionally, the stirring speed during alcohol precipitation is 10-600 rpm, optionally 40-500 rpm, further optionally 80-400 rpm or 300 rpm.

[0068] Optionally, the Moraceae plants are: *Morus multicaulis* Perrott., *Morus alba* L., *Morus atropurpurea* Roxb., *Morus mizuho* Hotta, *Morus wittiorum* Hand Mazz., *Morus laevigata* Wall., *Morus nigra* Linn., *Morus cathayana* Hemsi., *Morus serrata* Roxb., *Morus mongolica* Schneid., *Morus bombycis* Koidz., *Morus notabilis* Schneid., *Morus nigriformis* Koidz., *Morus yunnanensis* Koidz., *Morus australis* Poir., and *Morus mongolica(Bur.)Schneid var. diabolica*. (Koidz.), large-leaved mulberry, weeping mulberry (Morus alba var. Pendula Dippel), white-veined mulberry, and mulberry varieties bred from the above mulberry species, and hybrid mulberry varieties selected from the above mulberry species within or between species;

[0069] Optionally, the mulberry plant is selected from one or more of Guangdong mulberry, Lu mulberry, white mulberry, fine-toothed mulberry, mountain mulberry or hybrid mulberry, and the hybrid mulberry can be selected as Yue Sang 11, Gui Sang You 62 or Sang Te You 2. The leaves, roots, branches, bark, buds, stems, fruits, etc. of the mulberry plant can be used, and mulberry branches, mulberry leaves or mulberry bark can be used.

[0070] In this invention, the mulberry extract can be selected as mulberry branch extract, mulberry bark extract, mulberry leaf extract or a mixture thereof.

[0071] Optionally, the mulberry plant is crudely extracted using solvents such as alcohol-water, water, alkaline aqueous solution or acidic aqueous solution. During extraction, it is preferable to pulverize the plant and add it to a solvent for thermal extraction. Optionally, the extraction is repeated 1-3 times, and the extraction time is preferably 0.5-3 hours each time, and more preferably 1-3 hours each time.

[0072] In a preferred embodiment, the pulverized plant can be added to an extraction tank for extraction.

[0073] Preferably, the extract is filtered to remove insoluble matter, yielding a crude plant extract.

[0074] In one embodiment, the mulberry extract is prepared according to the following steps: pulverize mulberry plants, extract by heating and reflux with water and / or alcohol solution or acidic water, the solvent volume being 3-20 times (optionally 4-15 times) of the original medicinal material, repeat the extraction 1-3 times (extraction time can be 0.5-3h each time, further optionally 1-3h each time), combine the extracts, concentrate, load onto a cation exchange resin, elute with 0.2-3N ammonia water, load the eluent onto anion exchange resin, collect the non-adsorbed portion, add ethanol, precipitate to remove impurities, concentrate and / or dry to obtain the extract.

[0075] In one embodiment, the mulberry extract is prepared according to the following steps: pulverize the mulberry plant, extract it by heating and reflux with water and / or alcohol solution or acidic water, the solvent volume is 3-20 times (preferably 4-15 times) of the original medicinal material, repeat the extraction 1-3 times (the extraction time can be selected as 0.5-3h each time, and further selected as 1-3h each time), combine the extracts, concentrate, load onto a cation exchange resin, elute with 0.2-3N ammonia water, load the eluent onto anion exchange resin, collect the non-adsorbed part, concentrate and / or dry to obtain the extract.

[0076] In one embodiment, the mulberry extract is prepared according to the following steps: pulverizing mulberry plants, extracting them by heating and reflux with water and / or alcohol solution or acidic water, the solvent volume being 3-20 times (optionally 4-15 times, further optionally 4-12 times) of the original medicinal material, repeating the extraction 1-3 times (extraction time optionally 0.5-3 h each time, further optionally 1-3 h each time), combining the extracts, concentrating, loading onto a cation exchange resin, eluting with 0.2-3N ammonia water, concentrating and / or drying the eluent to obtain the extract.

[0077] Optionally, after packing the cation exchange resin column, activation is performed in the order of washing with acidic solution, washing with alkaline solution, and washing with acidic solution again. Optionally, the alkaline solution is used to wash until the pH of the eluent is 8.0-9.5, preferably 8.5-9.5; optionally, the alkaline solution is selected from ammonia solution, sodium hydroxide solution, potassium hydroxide solution, or sodium carbonate solution; optionally, the concentration of the alkaline solution is 0.5-4 mol / L, preferably 1-2 mol / L. Optionally, the acidic solution is used to wash until the pH of the eluent is 3.0-7.0, preferably 4.5-6.5. Optionally, the acidic solution is selected from hydrochloric acid solution, phosphoric acid solution, or disodium hydrogen phosphate-citric acid buffer solution; optionally, the concentration of the acidic solution is 0.5-4 mol / L, preferably 1.5-2 mol / L. Optionally, after the final acidic solution wash, the cation exchange resin can be rinsed with 3-5 column volumes of deionized water.

[0078] Preferably, the cation exchange resin is selected from strongly acidic cation exchange resins, weakly acidic cation exchange resins, and...The cation exchange resin is a combination of one or more of the following strongly basic quaternary ammonium cation exchange resins. Optionally, the cation exchange resin is a combination of one or more of the following: type 732 strongly acidic styrene-based cation exchange resin, type 002SC strongly acidic styrene-based cation exchange resin, type 734 strongly acidic styrene-based cation exchange resin, type D001 macroporous strongly acidic styrene-based cation exchange resin, type D113 macroporous weakly acidic cation exchange resin, and type D254 macroporous strongly basic quaternary ammonium cation exchange resin. Preferably, the cation exchange resin is a combination of one or more of the following: type 732 strongly acidic styrene-based cation exchange resin, type 734 strongly acidic styrene-based cation exchange resin, type 002SC strongly acidic styrene-based cation exchange resin, type D113 macroporous weakly acidic cation exchange resin, and type D001 macroporous strongly acidic styrene-based cation exchange resin.

[0079] Optionally, the ratio of the amount of cation exchange resin to the weight of the plant raw material is 1:1-30 (optionally, 1:1-25, 1:2-20, 1:2-15, 1:2-10, 1:2-7, 1:2-3, 1:4-15).

[0080] After loading the crude plant extract onto the cation exchange resin, the loaded cation exchange resin is eluted with an eluent. Optionally, the eluent is a salt solution or alkaline solution containing cations, and may be one or more of sodium chloride, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonia, potassium chloride, and sodium hydroxide.

[0081] Optionally, the concentration of cations in the eluent is 0.04-5 mol / L (optionally 0.5-2.5 mol / L, 0.2-3 mol / L, further optionally 0.5-2.5 mol / L).

[0082] Optionally, the eluent flow rate is 1-15 BV / h (optionally 5-10 BV / h, further optionally 5-6 BV / h).

[0083] Optionally, the weight of the eluent used for cation exchange is 0.1-30 times the weight of the plant raw material, as specified in the specification (page 7 / 23, 13 CN 121015729 A), optionally 0.5-10 times the weight of the plant raw material, further optionally 1-10 times.

[0084] The collection starting point can be determined according to the pH of the cation exchange resin effluent. For example, when using an alkaline solution such as ammonia for elution, the eluent is collected when the pH of the cation exchange column effluent is >7, or the collection starting point of the effluent is determined according to the colorimetric or precipitation reaction; optionally, when the volume of the collected liquid reaches 0.1-10 times the weight of the plant raw material (further optionally, ...), ...When the concentration of the cation exchange resin reaches 0.6-10 times (or 0.2-5 times), collection is stopped, and the collected liquid is optionally purified by passing it through an anion exchange column. To improve the separation effect of the cation exchange resin, the cation exchange resin can be used for multiple separations, for example, 2-5 separations.

[0085] When purifying by passing it through an anion exchange column, optionally, after the anion exchange resin is packed into the column, activation is performed in the order of washing with alkaline solution, washing with acidic solution, and washing with alkaline solution again.

[0086] Optionally, the alkaline solution is used to wash until the pH of the eluent is 8.0-9.5, preferably pH 8.5-9.5;

[0087] Optionally, the alkaline solution is selected from ammonia solution, sodium hydroxide solution, potassium hydroxide solution, or sodium carbonate solution; optionally, the concentration of the alkaline solution is 0.5-4 mol / L, preferably 1-2 mol / L.

[0088] Optionally, the acidic solution is used to wash until the pH of the eluent is 3.0-7.0, preferably 3.5-6.5. Optionally, the acidic solution is selected from hydrochloric acid solution, phosphoric acid solution, and disodium hydrogen phosphate-citric acid buffer solution. Optionally, the concentration of the acidic solution is 0.5-4 mol / L, and optionally 1-2 mol / L.

[0089] Preferably, the anion exchange resin is selected from one or more combinations of strongly basic anion exchange resin, weakly basic anion exchange resin, or weakly acidic anion exchange resin. Optionally, the anion exchange resin is one or more combinations of 711 type strongly basic styrene-based anion exchange resin, 717 type strongly basic styrene-based anion exchange resin, D201 type macroporous strongly basic styrene-based anion exchange resin, D218 type macroporous strongly basic acrylic anion exchange resin, D301-G type macroporous weakly acidic styrene-based anion exchange resin, and D301 type macroporous weakly basic styrene-based anion exchange resin. Preferably, the anion exchange resin is one or more of the following: type 711 strong base styrene-based anion exchange resin, type 717 strong base styrene-based anion exchange resin, type D201 macroporous strong base styrene-based anion exchange resin, and type D218 macroporous strong base acrylic anion exchange resin. Preferably, the anion exchange resin is one or more of the following: type 711 strong base styrene-based anion exchange resin, type D201 macroporous strong base styrene-based anion exchange resin, and type D218 macroporous strong base acrylic anion exchange resin.

[0090] Optionally, the ratio of the amount of anion exchange resin to the weight of the plant raw material is 1:1-80 (optionally, 1:1-64, 1:1-32, 1:1-24, 1:5-16, 1:3).

[0091] Collection begins when the liquid flows out of the anion exchange resin. Optionally, collection should be stopped when the volume of the collected liquid reaches 0.05-10 times (optionally 0.1-5 times) the weight of the plant raw materials. Optionally, to improve the separation effect of the anion exchange resin, the anion exchange resin can be used for multiple separations, for example, 2-4 separations.

[0092] Optionally, the weight ratio of ethanol to plant raw materials used in the alcohol precipitation treatment is 1:4-600 (optionally 1:20-300, further optional 1:20-200, 1:20-50, 1:40, 1:80, 1:22, 1:200). During the alcohol precipitation treatment, the stirring speed is 10-600 rpm (optionally 40-500 rpm, 80-400 rpm, 300-400 rpm). The alcohol precipitation treatment time is 12-24 hours.

[0093] Further, before the alcohol precipitation treatment, the method further includes centrifuging or microfiltration to remove impurities from the anion exchange resin effluent, followed by a reverse ion permeation membrane concentration step. The specific gravity of the concentrated liquid can be 1.0-1.3, optionally 1.1-1.25. This specific gravity refers to the mass ratio of the concentrated liquid to water under the same volume conditions.

[0094] The concentration process of the present invention may include thermal concentration, nanofiltration membrane, reverse ion osmosis membrane concentration, and combinations thereof. Preferably, centrifugation, ultrafiltration membrane filtration, or microfiltration membrane filtration are performed to remove impurities before reverse ion osmosis membrane and nanofiltration membrane concentration.

[0095] Optionally, the drug may further include a pharmaceutically acceptable carrier. The carrier is an inactive ingredient that is non-toxic to the human body and conforms to the route of administration or method of administration. The carrier may be a solid or liquid excipient. Solid excipients, for example, include microcrystalline cellulose, mannitol, lactose, pregelatinized starch, low-substituted hydroxypropyl cellulose, crospovidone, sodium carboxymethyl starch, aspartame, calcium hydrogen phosphate, sodium lactate, poloxamer, sodium lauryl sulfate, sodium carboxymethyl cellulose, gelatin, xanthan gum, povidone, starch, magnesium stearate, sodium carboxymethyl starch, and talc; liquid excipients, for example, include water, ethanol, syrup, and glycerin.

[0096] The beneficial effects of the present invention are as follows:

[0097] The mulberry extract of the present invention can promote the proliferation and migration of vascular endothelial cells, inhibit apoptosis, promote angiogenesis, and protect vascular endothelial cells from AGEs-induced oxidative stress damage.

[0098] The mulberry extract of the present invention regulates endothelial cell function by activating the NRF2 / HO-1 / eNOS signaling pathway, including promoting the expression of NRF2 and HO-1, increasing the NO content of endothelial cells, and promoting the expression of vasoactive factors, including promoting the expression of CD31, VEGF-A, or eNOS.

[0099] The mulberry extract of the present invention can inhibit the adhesion of HUVECs to monocytes induced by high concentrations of glucose and TNF-α, and alleviate the local amplification of inflammatory response.

[0100] The mulberry extract of the present invention can improve endothelial cell functional damage induced by high insulin, increase the number of mitochondria in endothelial cells, improve mitochondrial damage, and improve vascular endothelial cell function.

[0101] Animal experiments show that the mulberry extract of the present invention can promote the healing of diabetic foot wounds, including promoting wound regeneration.It promotes cell regeneration and re-epithelialization, and promotes collagen production; it reduces cell apoptosis in the wound area, promotes cell proliferation, promotes angiogenesis or vascular remodeling and / or repair, and promotes the expression of VEGF-A, CD31 and / or eNOS. The mulberry extract of the present invention can alleviate diabetic atherosclerosis: reduce arterial plaque formation, improve arterial blood flow, and reduce the level of vascular endothelial adhesion factors, including P-selectin and / or E-selectin. Figure Description

[0102] Figure 1 shows the effect of different concentrations of SZ-A on the viability of HUVECs in Experiment 1. Statistical analysis was performed using GraphPad Prism 8. Data are expressed as mean ± SD, **P<0.01.

[0103] Figure 2 shows the effect of SZ-A on AGEs-induced functional damage of HUVECs in Experiment 1, wherein,

[0104] a and b are microscopic images of tube formation assay and quantitative analysis of tube length (n=3), respectively;

[0105] c and d are microscopic images of cell scratches observed at 0, 8, 16 and 24 h, and d, e and f are quantitative analysis of wound closure rate at 8, 16 and 24 h (n=5), respectively;

[0106] g and h are microscopic images and quantitative analysis of cell migration in Transwell assay (n=4), respectively;

[0107] i and j are flow cytometry results and quantitative analysis of apoptosis (n=3), respectively;

[0108] k and l are TUNEL staining images and quantitative analysis (n=4), respectively. *p<0.05, **p

[0109] <0.01, ***p<0.001.

[0110] Figure 3 shows the effect of SZ-A on the NRF2 signaling pathway in HUVECs treated with AGEs in Experiment 1.

[0111] Where a, b, and c are Western blotting and quantitative statistical diagrams of the relative levels of NRF2 and HO-1 proteins in HUVECs after SZ-A intervention and AGEs treatment, respectively (n=4), and d and e are immunofluorescence and fluorescence intensity statistical analysis diagrams of NRF2 in HUVECs after SZ-A intervention and AGEs treatment, respectively (n=4). *p<0.05, **p<0.01.

[0112] Figure 4 shows the effect of SZ-A on vasoactive factors in HUVECs treated with AGEs in Experiment 1.

[0113] Wherein a, b, c, and d are Western blotting and quantitative statistical graphs of the relative levels of CD31, VEGF-A, and eNOS proteins in HUVECs after SZ-A intervention and AGEs treatment, respectively (n=4); e and f are immunofluorescence and quantitative statistical graphs of NO levels in HUVECs determined by DAF-FMDA method (n=4); g, h, i, and j are immunofluorescence and statistical graphs of CD31 and VEGF-A, respectively.Figure A (n=3), k, l, m, n, o, p are the Western blotting diagrams and relative protein level statistics of NRF2, HO-1, CD31, VEGF-A and eNOS after 24h of intervention with or without ML385 (n=4), *p<0.05, **p<0.01, ***p<0.001, ns indicates no significant difference.

[0114] Figure 5 shows the effect of SZ-A on the adhesion of HUVECs to Thp-1 induced by high glucose and TNF-α in Experiment 2;

[0115] where a is a microscopic image of HUVECs adhering to green fluorescently labeled Thp-1, b is a quantitative statistical graph of fluorescence intensity in the same area (n=4), ** indicates P<0.01 compared with the model, *** indicates P<0.001 compared with the model.

[0116] Figure 6 shows the effect of SZ-A on apoptosis of HUVECs induced by high glucose and TNF-α in Experiment 2;

[0117] where a is a flow cytometry analysis diagram, b is a statistical graph of late apoptosis rate (n=4), and * indicates P<0.05 compared with the model.

[0118] Figure 7 shows the effect of SZ-A on scratch healing of HUVECs induced by high glucose and TNF-α in Experiment 2;

[0119] where a is a visual diagram of cell scratches under a microscope, and b is a statistical graph of the scratch healing ratio of each group (n=4). * indicates P<0.05 compared with the model, ** indicates P<0.01 compared with the model, and *** indicates P<0.001 compared with the model.

[0120] Figure 8 shows the effect of SZ-A on high insulin-induced mitochondrial damage in HUVECs in Experiment 3;

[0121] where a is a fluorescence microscopy image of mitochondria in each group of HUVECs, b is a statistical graph of fluorescence intensity in each group, and * indicates P<0.05 compared with the model.

[0122] Figure 9 shows the effect of SZ-A on wound healing in diabetic mice in Experiment 4;

[0123] Wherein a and b are TUNEL staining images and quantitative fluorescence intensity images of pancreatic tissue of mice on day 7 after STZ administration (n = 4, compared with C57, ***P<0.001), c and d are representative images and schematic diagrams of the wound healing process of mice in each group on days 0, 3, 5, 7 and 9 after surgery, e is a quantitative assessment image of wound healing rate (n = 10), f, g and h are H&E staining images and quantitative evaluation of the width of new epithelial gap and the degree of re-epithelialization represented by horizontal black lines (n = 3), i and j are Masson trichrome staining images and quantitative collagen deposition volume images (n = 3). Statistical analysis was performed using GraphPad Prism 8. Data are expressed as mean ± SD, **P<0.01, ***P<0.001, ns indicates no significant difference.

[0124] Figure 10 shows the effect of SZ-A on vascular function during wound healing in diabetic mice in Experiment 4;

[0125] where a and b are representative immunofluorescence images and quantitative analysis images of cell apoptosis in skin wounds on day 9 after SZ-A intervention (n=3), c and d are representative immunofluorescence images and quantitative analysis images of cell proliferation in skin wounds on day 9 after SZ-A intervention (n=3), e and f are immunofluorescence staining images and quantitative analysis images of CD31 labeled with Alexa Fluor 488 and α-SMA labeled with Alexa Fluor 555, DAPI staining of cell nuclei (n=3), g, h and i, j are representative immunofluorescence images and quantitative analysis images of VEGF-A and eNOS in skin wounds on day 9 after SZ-A intervention (n=3). Statistical analysis was performed using GraphPad Prism 8. Data are expressed as mean ± SD. *P<0.05, **P<0.01, ***P<0.001, ns indicates no significant difference.

[0126] Figure 11 shows the effect of SZ-A on a single high-dose STZ injection model of diabetes AS in Experiment Example 5;

[0127] where a is the weight change curve; b is the postprandial random blood glucose bar graph; c is the fasting blood glucose bar graph; d is the gross Oil Red O staining map of the artery after treatment; e is the gross Oil Red O staining area statistical bar graph of the artery; f is the Oil Red O staining map of the arterial root after treatment; g is the statistical graph of E-selectin in serum; h is the statistical graph of P-selectin in serum; * indicates P < 0.05 compared with the model; ** indicates P < 0.01 compared with the model; *** indicates P < 0.001 compared with the model.

[0128] Figure 12 shows the effect of SZ-A on a diabetic AS model induced by five small-dose STZ injections in Experimental Example 5; where a is the survival curve; b is the weight change curve; c is the gross Oil Red O staining map of the arteries after treatment; d is the statistical bar chart of the gross Oil Red O staining area of ​​the arteries; e is the bar chart of random blood glucose; f is the bar chart of glycated hemoglobin; g is the bar chart of peak blood flow in the ascending aorta; h is the bar chart of peak blood flow in the descending aorta; i is the statistical chart of E-selectin in serum; j is the statistical chart of P-selectin in serum; * indicates P < 0.05 compared to the model, ** indicates P < 0.01 compared to the model, and *** indicates P < 0.001 compared to the model. Detailed Embodiments

[0129] The present invention will be further described in detail below through examples. Through these exemplary descriptions, the features and advantages of the present invention will become clearer.

[0130] The technical term “exemplary” herein means “serving as an example, embodiment, or illustration.” Any embodiment illustrated herein as “exemplary” is not necessarily to be construed as superior to or better than other embodiments.

[0131] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0132] Preparation of mulberry extract

[0133] Example 1 of preparation of mulberry extract

[0134] Take 1000 kg of fresh mulberry branches (fine-toothed mulberry, Yue mulberry 11), crush them, add 4000 L of water, extract by heating and reflux for 2 h, combine the extracts, filter to remove insoluble matter, and obtain crude extract. The crude extract is concentrated by heat until the solid content reaches 4%, and kept at 50℃ as the loading solution for the cation exchange resin column.

[0135] Use 150 kg of D113 type macroporous weak acid styrene-propylene cation exchange resin to pack the column, wash with 2 mol / L hydrochloric acid solution until the pH of the eluent is 4.5; wash with 1 mol / L sodium hydroxide solution until the pH of the eluent is 8.5; wash with 2 mol / L hydrochloric acid solution until the pH of the eluent is 4.5; then wash with 5 column volumes of deionized water to complete the activation. The concentrated extract was loaded onto the column and then eluted with 1000 L of 2.5 mol / L ammonia at a rate of 6 BV / h. The eluent was collected when the pH of the cation exchange column eluent was >7. Collection was stopped when the eluent reached 900 L, and the collected eluent was directly purified by an anion exchange column.

[0136] 62.5 kg of D218 macroporous strong basic acrylic anion exchange resin was packed into the column. The column was washed with 1.5 mol / L sodium hydroxide solution until the pH of the eluent was 9.0; then washed with 1.5 mol / L hydrochloric acid solution until the pH of the eluent was 3.5; and then washed with 1.5 mol / L sodium hydroxide solution until the pH of the eluent was 9.0; this completed the activation. The collected cation exchange resin eluent was loaded onto the anion exchange resin, and the eluent was collected until the eluent reached 920 L.

[0137] The collected liquid was centrifuged to remove impurities and then concentrated by reverse ion permeation membrane. The specific gravity of the concentrated liquid was 1.25. It was transferred to an alcohol precipitation tank and 25L of anhydrous ethanol was added at 500rpm. After the ethanol was added, the stirring was stopped and the alcohol precipitation was carried out for 24h. The supernatant was taken and concentrated under reduced pressure to obtain mulberry twig extract (i.e., mulberry extract SZ-A).

[0138] In the mulberry twig extract, the mass percentage of alkaloids was 52%, the mass percentage of polysaccharides was 22%, the mass percentage of flavonoids was 0.8%, and the mass percentage of amino acids was 20%. Among the alkaloids, the content of 1-DNJ was 60%, FAG was 17%, and DAB was 15%.

[0139] Example 2 of preparation of mulberry extract

[0140] 10kg of fresh mulberry twigs (Sangtuo No. 2) were taken, crushed, and 150L of water was added in two portions. Each time, the extract was extracted by decoction for 3h. The extracts were combined and filtered to remove insoluble matter. The extract was thermally concentrated until the solids content reached 8%, then transferred to an alcohol precipitation tank. 2367.9 g of anhydrous ethanol (3 L) was added under stirring at 300 rpm. After the ethanol addition was complete, stirring was stopped, and the alcohol precipitation proceeded.After 24 hours, the supernatant was used as the loading solution for the cation exchange resin column. 5 kg of 002SC type strong acid styrene-based cation exchange resin was packed into the column, and the cation exchange resin was activated according to the method in Preparation Example 1. The concentrated alcohol-precipitated extract was loaded onto the column, and then eluted with 100 L of 5 mol / L potassium chloride at a rate of 5 BV / h. The eluent was monitored with 20% silicotungstic acid. Collection began when a white precipitate formed, and stopped when 25 L of the collected solution was reached. The collected solution was then directly purified by an anion exchange column.

[0141] 10 kg of 711 type strong base styrene-based anion exchange resin was packed into the column, and the anion exchange resin was activated according to the method in Preparation Example 1. The collected cation exchange resin eluent was loaded onto the anion exchange resin, and the eluent was collected until 15 L of eluent was reached. The collected solution was reloaded onto the cation exchange resin, and the separation was repeated twice more using cation exchange resin and anion exchange resin in sequence according to the above method.

[0142] The collected liquid obtained after three column separations was centrifuged to remove impurities and then concentrated by reverse ion permeation membrane. The specific gravity of the concentrated liquid was 1.25. It was transferred to an alcohol precipitation tank, and 125g of anhydrous ethanol was added at 1000rpm with a stirring paddle. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24h. The supernatant was collected and concentrated under reduced pressure to obtain the extract. Fresh mulberry bark and mulberry leaves (Sangtuo No. 2) were also extracted using the same extraction method and parameters as described above.

[0143] The obtained mulberry twig extract contained 98% alkaloids, 0.2% polysaccharides, 0.05% flavonoids, and 0% amino acids. Among the alkaloids, the content of 1-DNJ was 99%, FAG was 0.5%, and DAB was 0.4%.

[0144] The obtained mulberry bark extract contained 95% alkaloids, 2% polysaccharides, 0.1% flavonoids, and 1% amino acids. Among the alkaloids, 1-DNJ was 96%, FAG was 1.5%, and DAB was 1.4%.

[0145] The obtained mulberry leaf extract contained 90% alkaloids, 4% polysaccharides, 0.1% flavonoids, and 3% amino acids. Among the alkaloids, 1-DNJ was 91%, FAG was 3.1%, and DAB was 2.8%.

[0146] Example 3 of mulberry extract preparation

[0147] 1000 kg of fresh mulberry branches (Guangdong mulberry) were crushed, 11500 L of water was added, and the mixture was heated under reflux for 2 h. The extracts were combined, filtered to remove insoluble matter, and a crude extract was obtained. The crude extract was first centrifuged to remove impurities, and then concentrated using a reverse ion permeation membrane until the solid content reached 1% by mass, which was then used as the loading solution for the cation exchange resin column.

[0148] A 300kg column of D001 type macroporous strong acid styrene-based cation exchange resin was packed, and the cation exchange resin was activated according to the method of Preparation Example 1. The concentrated crude extract was loaded onto the column and eluted with 5000L of 0.04mol / L ammonium nitrate at a rate of 5 BV / h. The eluent was monitored with 20% silicotungstic acid. Collection began when a white precipitate was formed and stopped when 1000L of the collected liquid was collected.

[0149] The collected liquid obtained after cation exchange column separation was concentrated by nanofiltration membrane and concentrated under reduced pressure to obtain an extract paste.

[0150] The mulberry twig extract obtained contained 15% alkaloids, 20% polysaccharides, 7% flavonoids, and 45% amino acids. Among the alkaloids, the content of 1-DNJ was 55%, FAG was 23%, and DAB was 10%.

[0151] Example 4 of preparation of mulberry extract

[0152] Take 333 kg of dried mulberry branches (Yue Sang No. 11), crush them, add 4000 L of water, and extract twice by heating and reflux, refluxing for 1 h each time. Combine the extracts, filter, and concentrate the extract to 1 kg of raw drug / L.

[0153] Pack 150 kg of D113 type macroporous weak acid styrene-propylene cationic resin column, wash with 2 mol / L hydrochloric acid solution until the pH of the eluent is 4.5; wash with 1 mol / L sodium hydroxide solution until the pH of the eluent is 8.5; wash with 2 mol / L hydrochloric acid solution until the pH of the eluent is 4.5; then wash with 5 column volumes of deionized water to complete the activation. The concentrated extract was loaded onto the column and then eluted with 1000 L of 2.5 mol / L ammonia at a rate of 6 BV / h. The eluent was collected when the pH of the cation exchange column eluent was greater than 7. When the collected eluent reached 900 L, the collection was stopped, and the collected eluent was directly purified by an anion exchange column.

[0154] A 125 kg column of D218 macroporous strong basic acrylic anion exchange resin was packed. The column was washed with 1.5 mol / L sodium hydroxide solution until the pH of the eluent was 9.0; then washed with 1.5 mol / L hydrochloric acid solution until the pH of the eluent was 3.5; and then washed with 1.5 mol / L sodium hydroxide solution until the pH of the eluent was 9.0; this completed the activation. The collected cation exchange resin eluent was loaded onto the anion exchange resin, and the eluent with a pH greater than 8 was collected until the eluent reached 870 L. Instructions for Use, pages 12 / 23, CN 121015729 A

[0155] The collected liquid obtained after separation by anion exchange column was filtered through a microfiltration membrane to remove impurities and then concentrated using a reverse ion permeation membrane. The specific gravity of the concentrated liquid was 1.1. It was transferred to an alcohol precipitation tank, and 15 kg of anhydrous ethanol was added at 400 rpm with a stirring paddle. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain mulberry twig extract. Sample content: biologicalThe alkali content is 80% by mass, the polysaccharide content is 5% by mass, the flavonoid content is 0.1% by mass, and the amino acid content is 4% by mass. In the alkaloids, the content of 1-DNJ is 75%, FAG is 12%, and DAB is 10%.

[0156] Example 5 of preparation of mulberry extract

[0157] Take 400 kg of dried mulberry branches (Yue Sang No. 11), crush them, add 4000 L of water, and extract twice by heating and reflux, refluxing for 1 h each time. Combine the extracts, filter, and concentrate the extract to 1 kg of raw drug / L.

[0158] Pack 62.5 kg of D218 type macroporous strong basic acrylic anion exchange resin column, wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluent is 9.0; wash with 1.5 mol / L hydrochloric acid solution until the pH of the eluent is 3.5; wash with 1.5 mol / L sodium hydroxide solution until the pH of the eluent is 9.0; complete the activation. The collected extract concentrate was loaded onto anion exchange resin, and 40 L of the eluent was collected.

[0159] The collected liquid obtained after separation by anion exchange column was filtered by microfiltration membrane to remove impurities and then concentrated by reverse ion exchange membrane. It was further concentrated and dried under reduced pressure to obtain mulberry twig extract. Sample content: alkaloid mass percentage of 3%, polysaccharide mass percentage of 70%, flavonoid mass percentage of 10%, amino acid mass percentage of 10%. Among the alkaloids, the content of 1-DNJ was 68%, FAG was 17%, and DAB was 8%.

[0160] Example 6 of preparation of mulberry extract

[0161] 1500 kg of fresh mulberry twigs (fine-toothed mulberry, Yue mulberry 11) were taken, crushed, and 6000 L of water were added. The extract was extracted by heating and reflux for 2 h. The extracts were combined, filtered to remove insoluble matter, and crude extract was obtained. The crude extract was concentrated by heat to the solid mass percentage of 4% and kept at 50 °C as the loading solution for cation exchange resin column.

[0162] A 100kg column of D113 macroporous weakly acidic styrene-propylene cation exchange resin was packed. The column was washed with 2mol / L hydrochloric acid solution until the pH of the eluent was 4.5; then washed with 1mol / L sodium hydroxide solution until the pH of the eluent was 8.5; then washed with 2mol / L hydrochloric acid solution until the pH of the eluent was 4.5; and finally rinsed with 5 column volumes of deionized water to complete the activation. The concentrated extract was loaded onto the column, and then eluted with 1000L of 2.5mol / L ammonia water at a rate of 6 BV / h. The eluent was collected when the pH of the cation exchange column effluent was >7. When the collected eluent reached 900L, the collection was stopped, and the collected eluent was directly purified by an anion exchange column.

[0163] A column was packed with 62.5 kg of D218 type macroporous strong basic acrylic anion exchange resin. The column was washed with 1.5 mol / L sodium hydroxide solution until the pH of the eluent was 9.0; then washed with 1.5 mol / L hydrochloric acid solution until the pH of the eluent was 3.5; 1.5 mol / L sodium hydroxide solution...Wash with sodium solution until the pH of the eluent is 9.0; activation is complete. Load the collected cation exchange resin eluent onto the anion exchange resin, and collect the eluent until it reaches 870 L. Concentrate the eluent under reduced pressure to obtain mulberry twig extract, wherein the mass percentage of alkaloids is 30%, the mass percentage of polysaccharides is 35%, the mass percentage of flavonoids is 2%, and the mass percentage of amino acids is 25%. Among the alkaloids, the content of 1-DNJ is 62%, FAG is 20%, and DAB is 13%.

[0164] Example 7 of preparation of mulberry extract

[0165] Take 1000 kg of fresh mulberry twigs (Fine-toothed Mulberry Yue Sang No. 11), crush them, add 4000 L of water, and extract by heating and reflux for 2 h. Combine the extracts, filter to remove insoluble matter, and obtain crude extract. Concentrate the crude extract by heat until the mass percentage of solids reaches 4%, and keep it at 50℃ as the loading solution for the cation exchange resin column.

[0166] A 100kg column of D113 macroporous weakly acidic styrene-propylene cation exchange resin was packed. The column was washed with 2mol / L hydrochloric acid solution until the pH of the eluent was 4.5; then washed with 1mol / L sodium hydroxide solution until the pH of the eluent was 8.5; then washed with 2mol / L hydrochloric acid solution until the pH of the eluent was 4.5; and finally rinsed with 5 column volumes of deionized water to complete the activation. The concentrated extract was loaded onto the column, and then eluted with 1000L of 2.5mol / L ammonia water at a rate of 6 BV / h. When the pH of the cation exchange column eluent was >7, the eluent was collected. When the collected liquid reached 900L, the collection was stopped, and the collected liquid was directly purified by passing it through an anion exchange column.

[0167] A column was packed with 62.5 kg of D218 type macroporous strong basic acrylic anion exchange resin. The column was washed with 1.5 mol / L sodium hydroxide solution until the pH of the eluent reached 9.0; then with 1.5 mol / L hydrochloric acid solution until the pH of the eluent reached 3.5; and finally with 1.5 mol / L sodium hydroxide solution until the pH of the eluent reached 9.0. Activation was then complete. The collected cation exchange resin eluent was loaded onto the anion exchange resin, and the eluent was collected until it reached 870 L. The eluent was concentrated under reduced pressure to obtain mulberry twig extract, which contained 40% alkaloids, 25% polysaccharides, 0.5% flavonoids, and 25% amino acids. Among the alkaloids, 1-DNJ accounted for 57%, FAG for 24%, and DAB for 16%.

[0168] Example 8 of the preparation of mulberry extract

[0169] 333 kg of dried mulberry branches (Yue Sang No. 11) were taken, crushed, and 4000 L of water were added. The extract was extracted twice by heating and reflux, with each reflux lasting 1 h. The extracts were combined, filtered, and concentrated to 1 kg of crude drug per L.

[0170] A 150 kg column of D113 macroporous weakly acidic styrene-propylene cation exchange resin was packed. The column was washed with 2 mol / L hydrochloric acid until the eluent pH reached 4.5; then with 1 mol / L sodium hydroxide solution until the eluent pH reached 8.5; and finally with 2 mol / L hydrochloric acid until the eluent pH reached 4.5. Activation was then achieved by rinsing with 5 column volumes of deionized water. The concentrated extract was loaded onto the column, followed by elution with 1000 L of 2.5 mol / L ammonia solution at a rate of 6 BV / h. The eluent was collected when the pH of the cation exchange column eluent was greater than 7. Collection was stopped when 900 L of eluent was collected, and the collected eluent was directly purified using an anion exchange column.

[0171] A column was packed with 62.5 kg of D218 type macroporous strong basic acrylic anion exchange resin. The column was washed with 1.5 mol / L sodium hydroxide solution until the pH of the eluent was 9.0; then with 1.5 mol / L hydrochloric acid solution until the pH of the eluent was 3.5; and finally with 1.5 mol / L sodium hydroxide solution until the pH of the eluent was 9.0. Activation was then completed. The collected cation exchange resin eluent was loaded onto the anion exchange resin, and the eluent with a pH greater than 8 was collected until the eluent reached 870 L.

[0172] The collected liquid obtained after separation by the anion exchange column was filtered through a microfiltration membrane to remove impurities and then concentrated using a reverse ion permeation membrane. The specific gravity of the concentrated liquid was 1.1. It was transferred to an alcohol precipitation tank, and 15 kg of anhydrous ethanol was added at 400 rpm. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 h. The supernatant was collected and concentrated under reduced pressure to obtain mulberry twig extract. Sample content: The mass percentage of alkaloids was 63%, the mass percentage of polysaccharides was 23%, the mass percentage of flavonoids was 1%, and the mass percentage of amino acids was 5%.

[0173] Among the alkaloids, the content of 1-DNJ was 61.9%, FAG was 16.6%, and DAB was 11.1%.

[0174] Example 9 of preparation of mulberry extract

[0175] 1000 kg of fresh mulberry branches (Yue Sang No. 11) were taken, crushed, and 4000 L of water were added. The extract was extracted by heating and reflux for 2 h. The extracts were combined, filtered to remove insoluble matter, and crude extract was obtained. The crude extract was concentrated by heat until the solid content reached 4%, and kept at 50 °C as the loading solution for cation exchange resin column.

[0176] A 120 kg column of D113 macroporous weakly acidic styrene-propylene cation exchange resin was packed. The column was washed with 2 mol / L hydrochloric acid solution until the pH of the eluent was 4.5; then washed with 1 mol / L sodium hydroxide solution until the pH of the eluent was 8.5; then washed with 2 mol / L hydrochloric acid solution until the pH of the eluent was 4.5; and finally rinsed with 5 column volumes of deionized water to complete the activation. The concentrated extract was loaded onto the column and then eluted with 1000 L of 2.5 mol / L ammonia solution at a rate of 6 BV / h. The eluent was collected when the pH of the cation exchange column effluent was >7. When the collected eluent reached 900 L, the collection was stopped, and the collected eluent was directly purified by passing it through an anion exchange column.

[0177] A 45kg column of D218 macroporous strong basic acrylic anion exchange resin was packed. The column was washed with 1.5mol / L sodium hydroxide solution until the pH of the eluent was 9.0; then with 1.5mol / L hydrochloric acid solution until the pH of the eluent was 3.5; and finally with 1.5mol / L sodium hydroxide solution until the pH of the eluent was 9.0. Activation was then complete. The collected cation exchange resin eluent was loaded onto the anion exchange resin collection solution until the eluent reached 870L.

[0178] The collected solution after anion exchange column separation was filtered through a microfiltration membrane to remove impurities and then concentrated using a reverse ion exchange membrane. The concentrated liquid had a specific gravity of 1.1. It was transferred to an alcohol precipitation tank, and 15kg of anhydrous ethanol was added with a stirrer at 300rpm. After the ethanol was added, stirring was stopped, and the alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain the extract. Sample content: 70% by mass of alkaloids, 20% by mass of polysaccharides, 0.6% by mass of flavonoids, and 5% by mass of amino acids.

[0179] Among the alkaloids, the content of 1-DNJ is 70%, FAG is 13%, and DAB is 10%.

[0180] Example 10 of the preparation of mulberry extract

[0181] 80 kg of fresh mulberry branches (Guisangyou 62) were taken, crushed, and 1000 L of water were added. The extract was extracted by heating and reflux for 2 h. The extracts were combined, filtered to remove insoluble matter, and crude extract was obtained. The crude extract was concentrated by heat until the solid content reached 4%, and kept at 50 °C as the loading solution for the cation exchange resin column.

[0182] A 50kg column of D113 macroporous weakly acidic styrene-propylene cation exchange resin was packed. The column was washed with 2mol / L hydrochloric acid solution until the pH of the eluent was 4.5; then washed with 1mol / L sodium hydroxide solution until the pH of the eluent was 8.5; then washed with 2mol / L hydrochloric acid solution until the pH of the eluent was 4.5; and finally rinsed with 5 column volumes of deionized water to complete the activation. The concentrated extract was loaded onto the column and then eluted with 600L of 2.5mol / L ammonia solution at a rate of 6 BV / h. The eluent was collected when the pH of the cation exchange column effluent was >7. When the collected eluent reached 800L, the collection was stopped, and the collected eluent was directly purified by passing it through an anion exchange column.

[0183] A 65kg column of D218 macroporous strong-base acrylic anion exchange resin was packed. The column was washed with 1.5mol / L sodium hydroxide solution until the eluent pH reached 9.0; then with 1.5mol / L hydrochloric acid solution until the eluent pH reached 3.5; and finally with 1.5mol / L sodium hydroxide solution until the eluent pH reached 9.0. Activation was then complete. The collected cation exchange resin eluent was loaded onto the anion exchange resin, and the eluent was collected until 750L was reached. The collected eluent was then reloaded onto the cation exchange resin, and the process was repeated once more using both cation and anion exchange resins.

[0184] The collected liquid obtained after two separations was filtered through a microfiltration membrane to remove impurities and then concentrated using a reverse ion permeation membrane. The specific gravity of the concentrated liquid was 1.1. It was transferred to an alcohol precipitation tank, and 400g of anhydrous ethanol was added at 350rpm with a stirring paddle. After the ethanol was added, stirring was stopped, and alcohol precipitation was carried out for 24 hours. The supernatant was collected and concentrated under reduced pressure to obtain the extract. Sample content: alkaloids accounted for 90% by mass, polysaccharides accounted for 3% by mass, flavonoids accounted for 0.5% by mass, and amino acids accounted for 2% by mass.

[0185] Among the alkaloids, the content of 1-DNJ was 94%, FAG was 2%, and DAB was 1%.

[0186] Pharmacological test of mulberry extract

[0187] In the following test examples, "SZ-A" represents mulberry extract.

[0188] Cell Experiments

[0189] Experiment 1: Effect of Mulberry Extract on AGEs (Advanced Glycosylation End Products)-Induced Functional Impairment of HUVECs

[0190] Cell Culture and SZ-A Intervention

[0191] Human umbilical vein endothelial cells (HUVECs) were obtained from Procell (Wuhan, China). HUVECs were incubated in a CO2 incubator at 37°C and 5% humidity in 11.1 mM glucose 1640 medium (with 1% antibiotics (penicillin / streptomycin) and 10% fetal bovine serum). In subsequent experiments, HUVECs were treated with SZ-A and incubated with 2 μg / mL AGEs (bs-1158P, bioss) to simulate the diabetic microenvironment for 24 h. The following experiments used HUVECs from passages 3 to 6.

[0192] 1. Cell viability assay

[0193] HUVECs were cultured for 24 h in a medium containing different concentrations of SZ-A (mulberry extract of Preparation Example 1, calculated as alkaloids, with concentrations of 0.1, 0.25, 0.5, 0.75, 1, 1.5, and 2 mg / mL, respectively). Cell viability was then measured using a Cell Counting Kit-8 (CCK-8). The absorbance was read at 450 nm using a Synergy H1 microplate reader. The results are shown in Figure 1.

[0194] The results showed that SZ-A promoted the proliferation of HUVECs.

[0195] 2. Tube formation assay

[0196] HUVECs were pretreated with 1 mg / mL SZ-A (mulberry extract of Preparation Example 1, calculated as alkaloids) for 3 h. They were then cultured for 24 h with or without 2 μg / mL AGEs. The HUVECs without SZ-A intervention were designated the Ctrl group, those with SZ-A intervention were designated the SZ-A group, HUVECs without AGEs treatment were designated the Vehicle group, and HUVECs treated with AGEs were designated the AGEs group. For example, the SZ-A group within the AGEs treatment group consisted of HUVECs treated with 1 mg / mL...SZ-A pretreatment for 3 h, followed by incubation with 2 μg / mL AGEs for 24 h.

[0197] Tube formation experiments were performed using matrix gel (356234, Corning) to evaluate the angiogenesis potential of HUVECs. 70 μL of liquid gel was uniformly added to each well of a 48-well plate placed on ice, and then incubated at 37°C for 30 min to allow the gel to solidify. Subsequently, drug-treated HUVECs were implanted into the solidified gel, and tube formation was monitored and recorded under a microscope within 6 h. The results are shown in Figures 2a and 2b.

[0198] The results showed that, without SZ-A intervention (Ctrl), the tube length in the AGEs group was significantly shorter than that in the Vehicle group. After SZ-A intervention, both the Vehicle group and the AGEs group showed significantly longer tube lengths.

[0199] 3. Cell Migration

[0200] Cell Scratch Assay: HUVECs were seeded into 6-well plates and pretreated with 1 mg / mL SZ-A (mulberry extract from Preparation Example 1, calculated as alkaloids) for 3 h, followed by intervention with or without AGEs for 24 h. After intervention, the cells were scratched with the tip of a 20 μL pipette. The migration process of HUVECs was observed and recorded within 24 h using a Lionheart FX automated microscope (Biotek, USA), and the degree of wound closure was indicated by a black horizontal line. The results are shown in Figure 2c. Quantitative analysis of wound closure rate at 8 h, 16 h, and 24 h is shown in Figures 2d, e, and f.

[0201] The results showed that, without SZ-A treatment, compared with the Vehicle group, the wound closure rate of HUVECs was significantly reduced at 16h and 24h after AGEs treatment, and the cell migration ability was decreased; after SZ-A intervention, the wound closure rate of the Vehicle group and the AGEs group was significantly increased (Figure 2c, d, e, f), and the cell migration effect of SZ-A on AGEs-treated HUVECs was more obvious.

[0202] Transwell assay method: HUVECs were pretreated with 1mg / mL SZ-A (mulberry extract of Preparation Example 1, calculated as alkaloids) for 3h, and then intervened with or without AGEs for 24h. After intervention, cells were seeded in the upper chamber of a Transwell plate (3422, Corning) at 4 × 10⁵ / mL per 100 μL serum-free medium, and 10% fetal bovine serum medium was added to the lower chamber. After incubation at 37°C for 24 h, the Transwell plate was removed, fixed with 4% paraformaldehyde for 15 min, and then stained with Coomassie brilliant blue for 15 min. The chamber was cleaned with PBS, wiped clean with cotton swabs, and dried. Then, it was observed, photographed, and counted using an inverted microscope. The results are shown in Figures 2g and h.

[0203] The results showed that after SZ-A intervention, the number of cells migrating in the Vehicle group and the AGEs group increased.

[0204] 4. Apoptosis assay

[0205] Flow cytometry: 1 mg / mLHUVECs were pretreated with SZ-A (mulberry extract from Preparation Example 1, calculated as alkaloids) for 3 h, followed by intervention with or without AGEs for 24 h. All steps were performed according to the instructions provided with the apoptosis analysis kit (FXP023, 4Abio). The apoptosis level in each group was assessed using the TUNEL apoptosis detection kit (KGA702, KeyGen Biotech).

[0206] The results showed that after SZ-A intervention, apoptotic cells in HUVECs (AGEs group) under simulated hyperglycemic pathological conditions were significantly reduced, and SZ-A showed a significant anti-apoptotic effect (Fig. 2i, j). TUNEL staining results showed that apoptosis in the AGEs group was inhibited after SZ-A intervention (Fig. 2k, l).

[0207] 5. Effect of SZ-A on vasoactive factors in HUVECs treated with AGEs

[0208] Protein separation and Western blotting analysis: Proteins were extracted from HUVECs cells using RIPA lysis buffer with added protease (sc-364162, Santa Cruz Biotechnology) and phosphatase inhibitor (B15001, Selleck). Total protein content was quantified using a BCA protein assay kit (ZJ102, Epizyme). The protein was separated by polyacrylamide gel electrophoresis (see instructions page 16 / 23, CN 121015729 A), then transferred to a PVDF membrane (Millipore, Billeria). The membrane was then blocked in TBST with 5% milk for 1 hour and incubated overnight at 4°C with primary antibodies against NRF2 (AF0639, Affinity Bioscience), HO-1 (T55113, Abmart), eNOS (345227, Zen Bio), CD31 (R10021, Zen Bio), VEGF-A (WL00009b, WanLeiBio), and β-Tubulin (10094-1-AP, Proteintech). After washing three times with TBST buffer, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (31430, Thermo Fisher Scientific). The antigen was then visualized using a chemiluminescence assay kit (P2300, NCM Biotech). Immunoreactive bands were visualized using a ChemiDoc Touch (Bio-Rad, USA) chemiluminescence imaging system and analyzed using ImageJ.

[0209] Endogenous nitric oxide (NO) assay: As a key regulator of NO synthesis, the importance of eNOS in vascular function regulation is widely recognized. Further analysis was performed using DAF-FM...The NO accumulation in HUVECs was detected using a DA kit (S0019S, Beyotime). HUVECs were pretreated with 1 mg / mL SZ-A (mulberry extract from Preparation Example 1, calculated as alkaloids) for 3 h, and then exposed to AGEs or blank control conditions for 24 h. After PBS recovery and washing, DAF-FM DA was added to a final concentration of 2.5 μM. The samples were then incubated at 37 °C in the dark for 20 min, followed by washing three times with PBS. The samples were visualized using a fluorescence microscope and quantitatively analyzed using ImageJ.

[0210] The levels of NRF2, CD31, and VEGF-A were detected using immunofluorescence.

[0211] Further, HUVECs cells were pretreated with 1 mg / mL SZ-A (mulberry extract from Preparation Example 1, calculated as alkaloids) for 3 hours, and then intervened with ML385 or without ML385 (2 μg / mL) for 24 hours. The expression levels of NRF2, HO-1, CD31, VEGF-A and eNOS in HUVECs were detected by Western blot. ML385 is an NRF2 inhibitor.

[0212] Experimental results:

[0213] The results in Figure 3 show that under simulated high glucose pathological conditions (HUVECs treated with AGEs), compared with the blank control group (Ctrl group), SZ-A can promote the expression of NRF2 (Figure 3a, b), which is consistent with the results of immunofluorescence quantitative analysis (Figure 3d, e); and SZ-A can upregulate the expression level of downstream effector molecule HO-1 (heme oxygenase 1) (Figure 3a, c). This indicates that SZ-A can regulate the NRF2 / HO-1 signaling pathway.

[0214] Figure 4 shows that SZ-A can significantly enhance the levels of CD31 (also known as platelet-endothelial cell adhesion factor-1) and VEGF-A (vascular endothelial growth factor-A), and significantly upregulate the expression of eNOS (endothelial nitric oxide synthase) (Figure 4a, b, c, d). After SZ-A intervention, the NO content in the Vehicle group and AGEs group was significantly increased (Figure 4e, f), and the expression levels of CD31 and VEGF-A were significantly enhanced (Figure 4g, h, i, j). In simulated hyperglycemic pathological conditions (HUVECs treated with AGEs), in untreated HUVECs (Vehicle group), administration of SZ-A significantly promoted the expression of NRF2, HO-1, CD31, VEGF-A, and eNOS; in HUVECs treated with ML385 (ML385 group), the promoting effect of SZ-A on the expression of NRF2, HO-1, CD31, VEGF-A, and eNOS was inhibited (Figure 4k, l, m, n, o, p).

[0215] The above results indicate that the endothelial cell protection effect mediated by SZ-A is closely related to the NRF2 / HO-1 / eNOS signaling pathway.It can improve endothelial cell function by promoting the expression of vasoactive-related factors (such as eNOS, CD31 and VEGF-A).

[0216] In summary, SZ-A can effectively inhibit endothelial cell function damage caused by AGEs.

[0217] Experimental Example 2: Effect of mulberry extract on HUVECs damage induced by high concentration of glucose and TNF-α

[0218] Inflammation and high glucose can cause the secretion of endothelial cell adhesion factors, causing the adhesion of monocytes and macrophages, resulting in the amplification of local endothelial cell inflammatory response. By constructing an in vitro model of HUVECs damage induced by high concentration of glucose (referred to as high glucose) and tumor necrosis factor-α (TNF-α), the effect of mulberry extract on HUVECs damage was investigated.

[0219] 1. Cell Adhesion

[0220] Test Cells: Human Umbilical Vein Endothelial Cells (HUVEC) and Human Monocytic Leukemia Cells (THP-1) (HUVEC was provided by the Union Cell Bank of the Chinese Academy of Medical Sciences; THP-1 was purchased from Wuhan Pronosei Life Science Technology Co., Ltd.) Instruction Manual 17 / 23 Page 23 CN 121015729 A

[0221] Take HUVEC cells seeded in 10 mm2 culture dishes, digest them with 0.25%, collect the cells, centrifuge at 300×g for 5 min, discard the supernatant, resuspend the cells in complete culture medium (DMEM-H (cellmax CGM113.05) + 10% FBS (Sino Biological FBS FBS01)) and count them; dilute the resuspended HUVEC cells to a concentration of 105 cells / mL and seed them in 12-well plates; pretreat HUVEC cells with culture medium containing 50 mmol glucose and 20 ng / mL TNF-α, and add 500 μg / Mulberry extract SZ-A (prepared from Preparation Examples 3, 6, 8, and 10, calculated as alkaloids) was applied for 24 hours. After the cells reached confluence, the following pretreatment Thrp-1 was added.

[0222] Thp-1 pretreatment staining:

[0223] (1) Centrifuge Thp-1 cells at 300×g for 5 min, and resuspend the cell pellet in serum-free RPMI-1640 medium at a density of 5×106 cells / mL;

[0224] (2) Add CFSE fluorescent probe (Beyotime C1031) to Thp-1 cells under light-protected conditions to a final concentration of 5 μM (dissolved in DMSO to prepare a 5 mM stock solution);

[0225] (3) Incubate the Thp-1 cells containing the probe in a CO2 cell culture incubator for 15 min in the dark;

[0226] (4) Add an equal volume of RPMI-1640 medium containing 20% ​​fetal bovine serum to the Thp-1 cells containing the probe to terminate the reaction for 10 min;

[0227] (5) Centrifuge the CFSE-labeled Thp-1 cells at 300×g for 5 min, resuspend in PBS, centrifuge again, and discard the supernatant;

[0228] (6) Resuspend CFSE-labeled THP-1 cells in RPMI-1640 medium containing 10% fetal bovine serum to achieve a cell density of 1×10⁶ cells / mL;

[0229] (7) Add the above-labeled THP-1 cells to HUVECs cells (20 μL of labeled THP-1 cells to 1 mL of endothelial cell culture medium) and culture them together in the dark for 6 h.

[0230] Wash away THP-1 cells that have not bound to HUVECs with PBS, then add an appropriate amount of PBS and observe the adhesion of HUVECs and THP-1 cells under a fluorescence microscope. The number of green fluorescent cells was used for evaluation, and the results are shown in Figure 5.

[0231] The results showed that HUVECs (model group) treated with high concentrations of glucose and TNF-α had the highest number of adhesions to Thp-1. After treatment with SZ-A, all treatment groups could inhibit the adhesion of HUVECs to Thp-1 to a certain extent, indicating that mulberry extract can alleviate local inflammation of vascular endothelium by inhibiting the adhesion between monocytes and endothelial cells.

[0232] 2. Apoptosis

[0233] HUVEC cells seeded in 10 mm2 culture dishes were digested with 0.25% and collected. The cells were centrifuged at 300×g for 5 min, the supernatant was discarded, and the cells were resuspended in complete culture medium and counted. The resuspended HUVEC cells were diluted to a concentration of 105 cells / mL, seeded in 6-well plates and cultured for 24 h. Then, culture medium containing 50 mmol glucose and 20 ng / mL TNF-α was added, and 500 μg / mL mulberry extract (prepared from preparation examples 6, 8, and 10, calculated as alkaloids) was added for 24 h. Then, the apoptosis rate was detected by flow cytometry. Annexin V fluorescent probe labeling was used, and cells positive for both FITC and PI were used as apoptotic cells. The differences between groups were compared by one-way ANOVA. The flow cytometry results of HUVEC cell apoptosis are shown in Figure 6, and the late apoptosis rate is shown in Table 1.

[0234] Table 1 Results of late apoptosis rate of mulberry extract on high glucose and TNF-α induced HUVECs (n=4)

[0235] Group Late apoptosis rate (%) Control 2.03 Model 4.43 Preparation Example 6 Mulberry extract 3.87 Preparation Example 8 Mulberry extract 3.78 Preparation Example 10 Mulberry extract 3.55

[0236] The results show that the mulberry extract of the present invention can inhibit HUVECs cell apoptosis to a certain extent. Instructions for Use, Pages 18 / 23, 24, CN 121015729 A

[0237] 3. Cell Scratch Assay

[0238] HUVEC cells seeded in 10 mm² culture dishes were digested with 0.25% and collected. The cells were centrifuged at 300 × g for 5 min, the supernatant was discarded, and the cells were resuspended in complete culture medium and counted. The resuspended HUVEC cells were diluted to a concentration of 10⁵ cells / mL and inoculated.The cells were seeded in 12-well plates. After the cells reached confluence, a vertical line was drawn at the bottom of the well plate using a 200 μL pipette tip.

[0239] The cells were cultured in a medium containing 50 mmol glucose and 20 ng / mL TNF-α, and then treated with 500 μg / mL mulberry extract (prepared from Preparation Examples 3, 6, 8, and 10, calculated as alkaloids). The cells were washed three times with PBS for 5 min each time, and cell migration was recorded at 0 h, 12 h, and 24 h under an inverted microscope.

[0240] The scratch healing rate of HUVECs at 24 h was calculated, i.e., the migration rate. Cell migration rate = (L0 – Lt) / L0 × 100%. L0: scratch distance at 0 h; Lt: scratch distance at the set detection time. The images were processed using the built-in microscopy software gene5. The distance was measured at three random locations for each scratch using the fluorescence microscope length measurement function, and the average value was calculated as the scratch distance for this field of view. The data were compared between groups using one-way ANOVA. The results are shown in Figure 7. The ability of mulberry extract to promote endothelial cell migration and proliferation was evaluated. The specific scratch healing ratio is shown in Table 2 below.

[0241] Table 2 Results of scratch healing ratio of mulberry extract on high glucose and TNF-α induced HUVEC damage (n=4)

[0242]

[0243] The results show that the mulberry extract of the present invention can significantly increase the migration rate of HUVEC cells to a certain extent and promote scratch healing.

[0244] Experimental Example 3: Effect of mulberry extract on high insulin-induced HUVEC cell damage

[0245] Cell culture: Human umbilical vein endothelial cells (HUVEC) were cultured in DMEM medium (10% FBS + 1% double antibiotics).

[0246] Cell passage: Digested with 0.25% trypsin, seeded at a density of 105 / mL in 12-well plates, and cultured in a cell culture incubator at 37℃ and 5% CO2. The culture medium was changed every 2-3 days. Once the cells occupied about 80% of the field of view, they could be passaged again.

[0247] Model construction: After passage, HUVEC cells were cultured overnight and then randomly divided into a normal control group, a model group, and a mulberry extract SZ-A group, for a total of 6 groups. The normal control group was given DMEM medium (10% FBS + 1% penicillin antibody), the model group (10% FBS + 1% penicillin antibody + 40U insulin), and the SZ-A group (mulberry extract from Preparation Example 8, calculated as alkaloids, with a concentration of 100, 200, 300, and 400 μg / mL) and the corresponding concentration of SZ-A group (10% FBS + 1% penicillin antibody + 40U insulin + corresponding concentration of SZ-A). After culturing for 24 h, mitochondria-specific fluorescence staining was performed using Mito-Tracker Green.

[0248] When the cells reached a certain density in the cell culture plate or culture dish, the cell culture medium was removed, and the prepared Mito-Tracker Green solution was added.After incubating the Green (mitochondrial green fluorescent probe) working solution at 37°C for 15-30 minutes, the Mito-Tracker Green working solution was removed, and fresh cell culture medium pre-warmed at 37°C was added. Observation under a fluorescence microscope showed that the mitochondria exhibited bright and strong fluorescent staining. The results are shown in Figure 8.

[0249] The results showed that compared with the model group, the SZ-A group significantly increased the number of mitochondria in endothelial cells and improved mitochondrial damage, suggesting that mulberry extract can significantly improve endothelial cell function.

[0250] Animal Experiments

[0251] Experiment 4: Effect of Mulberry Extract on Wound Healing in Diabetic Mice

[0252] Construction of Diabetic Mouse Model:

[0253] Male C57BL / 6J mice (purchased from Saiyi Biotechnology Co., Ltd. (Suzhou, China)) aged 6-8 weeks were housed in a rodent facility, given 12 hours of light and dark illumination, and free access to food and water. The animal research was approved by the Research Animal Ethics Committee of Chongqing University (COU-IACUC-RE-202308-005). Mice were fed a high-fat HFD diet (60% fat, D12492, Research Diets) for 4 weeks, followed by intraperitoneal injection of 35 mg / kg streptozotocin (STZ; S0130; Sigma-Aldrich), dissolved in citrate buffer (pH 4.2-4.5), once daily for 5 consecutive days. Blood glucose levels were continuously measured via tail vein sampling. Mice with a random blood glucose level ≥16.7 mmol / L three or more times after one week were classified as diabetic mice and used for the following experiments. On day 7, pancreatic tissue was collected from both STZ-treated and STZ-free mice. The TUNEL apoptosis assay kit (KGA702, KeyGen Biotech) was used to assess the apoptosis levels in each group and observe tissue damage.

[0254] Grouping and administration of experimental animals:

[0255] Diabetic mice were divided into a normal control (NC) group, an SZ-A-200 group, and an SZ-A-400 group (SZ-A was the mulberry extract of Preparation Example 1, and the dosage was 200 mg / kg and 400 mg / kg respectively, calculated as alkaloids). The mice were administered the drug for 15 consecutive days using a mixed feeding method (the drug was mixed into the HDF high-fat diet), and then 6 mm diameter wounds were created in each group.

[0256] Construction of the diabetic mouse skin wound model:

[0257] The model was created as follows: animals were anesthetized with isoflurane (RDW), and a sterile 6 mm cylindrical perforation was used to induce full-thickness wounds on both sides of the back. Observations were recorded for 9 days, with continuous administration during this period. Digital images were acquired on days 0, 3, 5, 7, and 9 post-surgery. ImageJ software was used to standardize and calibrate the wound area measurement and wound closure rate.

[0258] Mice were sacrificed on day 9, and wound tissue was collected using an 8mm skin biopsy well to analyze the expression of mRNA and protein during wound healing.

[0259] Immunofluorescence and morphological staining

[0260] The sections were fixed in 4% paraformaldehyde at 4°C for 16-24 h, embedded in paraffin, and sectioned to 5 μm. The paraffin sections were dewaxed, the antigen was extracted with sodium citrate, and then the sections were washed with PBS and incubated in blocking buffer. Subsequently, the sections were incubated overnight at 4°C with primary antibodies of eNOS (345227, Zen Bio), CD31 (R10021, Zen Bio), α-SMA (55135-I-AP, Proteintech), and VEGF-A (WL00009b, WanLeiBio). The sections were incubated with secondary antibodies (Thermo Fisher) conjugated with Alexa Fluor 488 and Alexa Fluor 555 and then stained with immunofluorescence. The fluorescence was then maintained during tissue imaging using DAPI staining solution (C1005, Beyotime) and anti-fluorescence quenching mounting agent (ab104135, Abcam). Collagen was quantified in skin areas stained with Masson's trichrome staining kit (G1346 Solarbio) using ImageJ software. Cells cultured on coverslips were fixed by treating with 4% paraformaldehyde PBS for 10 min. Subsequently, cells were washed in PBS and permeated with PBG-Triton (containing PBS, 0.4% fish skin gelatin, 0.5% bovine serum albumin, and 0.5% Triton X-100) for 1 h. Cells were then incubated with primary antibody overnight at 4°C. After washing with PBS, secondary antibody was applied and incubated at room temperature for 1 h. Coverslips were mounted on slides using a ThermoFisher Scientific extended Gold Antifade Mountant with DAPI. Imaging was performed using an APEXVIEWT MAPX100 All-in-One microscope (Olympus, Japan). Fluorescence images were quantitatively analyzed using ImageJ software.

[0261] Experimental Results:

[0262] 1. SZ-A can promote wound healing in diabetic mice.

[0263] Immunofluorescence images and analysis showed that, compared with normal mice, significant apoptosis occurred in the pancreatic tissue of mice 7 days after STZ administration, indicating that the pancreatic tissue was damaged (Fig. 9a, b).

[0264] In order to reduce wound contraction in rodent models and simulate the wound healing process of human skin tautness, silicone pads were applied to the wound to exert an anti-contraction effect, and the wound healing status was comprehensively evaluated. Fig. 9c, d show the changes in wound healing on days 0, 3, 5, 7 and 9 after oral administration of different concentrations of SZ-A to diabetic mice. The wound healing rate showed that...Compared with the NC group, SZ-A can more effectively promote the healing of diabetic wounds with increasing dosage (Fig. 9e). Quantitative analysis of wound length and horizontal black lines in HE staining images showed that the SZ-A group was significantly better than the NC group in terms of wound regeneration and re-epithelialization (Fig. 9f, g, h). Masson trichrome staining to measure collagen deposition showed that SZ-A had a stimulating effect on collagen production compared with the NC group (Fig. 9i, j).

[0265] The above results indicate that SZ-A can promote the healing of diabetic wounds.

[0266] 2. SZ-A can improve vascular function during the healing process of diabetic wounds

[0267] The results in Fig. 10a and b show that, compared with the NC group, on day 9 after SZ-A intervention, the SZ-A-200 group and the SZ-A-400 group significantly reduced cell apoptosis in the diabetic wound area. Figures 10c and 10d show that SZ-A intervention significantly upregulated the expression level of ki67 (a nuclear protein closely related to cell proliferation), indicating that SZ-A has a promoting effect on cell proliferation in diabetic wound areas.

[0268] Further, CD31 and α-SMA (α-smooth muscle actin) were used as indicators of endothelial cells and smooth muscle cells, respectively, to assess the degree of angiogenesis. Figures 10e and 10f show that SZ-A intervention promoted the angiogenesis process and showed a dose-gradient dependence, indicating its potential in promoting angiogenesis and tissue repair.

[0269] Further, the expression levels of VEGF-A and eNOS during the healing process of diabetic wounds after SZ-A intervention were evaluated. Figures 10g, h, i, and j show that SZ-A intervention significantly upregulated the expression levels of VEGF-A and eNOS and showed a dose-gradient dependence.

[0270] The above experimental results indicate that SZ-A intervention can enhance vascular remodeling and repair.

[0271] Experimental Example 5: Effect of Mulberry Extract on Diabetic Atherosclerosis (AS)

[0272] 1 Animal Model and Grouping

[0273] 1.1 Establishment and Grouping of Diabetic Atherosclerosis Animal Model (Single High-Dose STZ)

[0274] Eight-week-old SPF-grade C57 BL / 6J and ApoE- / - mice were acclimatized for one week. C57BL / 6J served as the control group and was fed a normal standard diet; ApoE- / - mice served as the modeling group and were fed HFHC (high-cholesterol diet, 40% fat, 1.25% cholesterol) for 5 weeks, and then injected intraperitoneally with 100 mg / kg STZ (streptozotocin). Blood glucose was measured on the 3rd day after injection. A blood glucose value ≥ 11 mmol / L indicated successful modeling and was used for subsequent experiments.

[0275] After successful modeling, mice were randomly grouped according to body weight and blood glucose level. The experimental groups and drug administration are shown in Table 3. Drug administration was carried out in the following manner for 6 consecutive weeks.

[0276] Table 3 Experimental grouping and administration of diabetic AS animal model (single high-dose STZ)Instructions for Use, Pages 21 / 23, 27, CN 121015729 A

[0277]

[0278] 1.2 Establishment and Grouping of Diabetic Atherosclerosis Animal Model (Five Small-Dose STZ)

[0279] Eight-week-old SPF-grade ApoE- / - mice were acclimatized for one week. The mice were divided into a control group and a model group according to their weight and blood glucose. The control group was fed a normal standard diet; the model group was fed HFHC (high-cholesterol diet, 40% fat, 1.25% cholesterol). After 6 weeks, STZ was injected intraperitoneally once a day at a dose of 50 mg / kg for 5 consecutive days. Blood glucose was measured on days 3, 6, and 9 after injection. A blood glucose value ≥16.7 mmol / L for three consecutive times was considered a successful model and was used for subsequent experiments.

[0280] After successful modeling, the mice were randomly grouped according to their weight and blood glucose value. The experimental groups and administration are shown in Table 4. The mice were administered the following drugs for 12 consecutive weeks.

[0281] Table 4 Grouping and administration of diabetic AS animal model (five small-dose STZ injections)

[0282]

[0283] 2 Experimental methods and results

[0284] 2.1 SZ-A effectively alleviates the disease progression of diabetic AS model after a single large-dose STZ injection

[0285] After three weeks of treatment, blood was collected from the tail tip, and random blood glucose was measured and recorded using a blood glucose meter. After treatment, the mice were fasted overnight, and blood was collected from the orbital veins after euthanasia. Fasting blood glucose was measured using a blood glucose meter, and serum was separated from the remaining whole blood. The levels of serum P-selectin and E-selectin were detected by ELISA, and the concentration was analyzed according to the standard curve. The whole body was perfused with pre-cooled physiological saline until the outflow was clear. The aorta was freed under a stereomicroscope and longitudinally cut. It was fixed with 4% paraformaldehyde, stained with Oil Red O working solution, and incubated. It was washed twice with 70% ethanol. The tissue was immersed in PBS and photographed. The percentage of plaque area was calculated. The experimental results are shown in Figure 11.

[0286] The results showed that, compared with the control group, the body weight of the STZ-injected model mice showed a decreasing trend (Fig. 11a). SZ-A treatment significantly reduced fasting blood glucose (Fig. 11c) and random blood glucose (Fig. 11b), with the high-dose SZ-A group showing comparable effects to the positive control group (22 / 23 pages, CN 121015729 A SGLT2i group). Furthermore, the arterial plaque area was significantly reduced in all SZ-A dose groups (Fig. 11d, e, f), with the high-dose SZ-A group showing better effects than the SGLT2i group. In addition, SZ-A significantly reduced the levels of E-selectin and P-selectin (Fig. 11g and h).

[0287] 2.2 SZ-A effectively alleviated the disease progression of a diabetic AS model after five low-dose STZ injections

[0288] After five weeks of treatment, blood was collected from the tail tip, and random blood glucose was measured and recorded using a blood glucose meter. Overnight fasting was observed after treatment.After feeding and anesthesia, arterial blood flow in mice was measured by ultrasound. The mice were then euthanized, and blood was collected from the orbital vein to measure glycated hemoglobin (HBA1c). The remaining serum was separated, and serum P-selectin and E-selectin levels were detected by ELISA, with concentration analysis based on a standard curve. The mice were perfused with pre-cooled physiological saline until the outflow was clear. The aorta was freed under a stereomicroscope and longitudinally cut. It was fixed with 4% paraformaldehyde, stained with Oil Red O working solution, and incubated. The tissue was washed twice with 70% ethanol. The tissue was immersed in PBS and photographed. The percentage of plaque area was calculated. The experimental results are shown in Figure 12.

[0289] The results showed that after STZ injection, the model animals died (Figure 12a) and their body weight decreased (Figure 12b). Compared with the model group, each dose of SZ-A significantly reduced random blood glucose (Figure 12e) and glycated hemoglobin (Figure 12f), while also reducing arterial plaque formation (Figure 12c, d). Compared with the model group, the SZ-A100 group showed an increasing trend in peak descending aortic blood flow (Figure 12h, P = 0.052), with mean ascending and descending aortic blood flow increasing by 21.79% and 15.73%, respectively, indicating that SZ-A has an effect on improving arterial blood flow. Furthermore, the SZ-A100 group significantly reduced the levels of P-selectin and E-selectin (Figures 12i and 12j). Instruction manual page 23 / 23, page 29, CN 121015729 A, Figure 1; Instruction manual figure 1 / 11, page 30, CN 121015729 A, page 2 / 11, page 31, CN 121015729 A, Figure 2; Instruction manual figure 3 / 11, page 32, CN 121015729 A, Figure 3; Instruction manual figure 4 / 11, page 33, CN 121015729 A, page 5 / 11, page 34, CN 121015729 A, Figure 4; Instruction manual figure 5; Instruction manual figure 6 / 11, page 35, CN 121015729 A, Figure 6; Instruction manual figure 7 / 11, page 36, CN 121015729 A, Figure 8; Instruction manual figure 8 / 11, page 37, CN 121015729 A, Figure 9; Instruction manual figure 10; Instruction manual figure 9 / 11, page 38, CN 121015729 A, Figure 11. Instruction manual, Figure 10 / 11, page 39, CN 121015729 A; Figure 12, Instruction manual, Figure 11 / 11, page 40, CN 121015729 A; Abstract: Abdominal ultrasound examination method.system and device USE OF MULBERRY EXTRACT IN PREPARATION OF MEDICAMENT FOR TREATING AND / OR PREVENTING DIABETIC VASCULAR COMPLICATIONS Use of a Morus extract, said Morus extract comprising alkaloids in an amount of more than 3% w / w, and / or comprising polysaccharides in an amount of no more than 70% w / w, and / or comprising flavonoids in an amount of no more than 10% w / w, and / or comprising amino acids in an amount of no more than 50% w / w, and / or other components, for the manufacture of a medicament for the treatment and / or prevention of vascular complications of diabetes. The ameliorating effect of Morus extract according to the present invention on vascular complications of diabetes is significant.

Claims

1. The use of mulberry extract in the preparation of medicaments for the treatment and / or prevention of diabetic vascular complications, characterized in that, Based on the total weight percentage of all components of the mulberry extract as 100%, the mulberry extract contains alkaloids at a weight content of 3% or more (optionally containing alkaloids at a weight content of 3-99%, further optionally containing alkaloids at a weight content of 15-99%, further optionally containing alkaloids at a weight content of 30-99%, further optionally containing alkaloids at a weight content of 40-99%, further optionally containing alkaloids at a weight content of 50-99%, further optionally containing alkaloids at a weight content of 60-99%). And / or contains polysaccharides with a weight content not exceeding 70% (optionally containing 0.2-70% polysaccharides, further optionally containing 0.2-50% polysaccharides, further optionally containing 0.2-35% polysaccharides, further optionally containing 0.2-25% polysaccharides, further optionally containing 0.2-23% polysaccharides, further optionally containing 20-25% polysaccharides). And / or contains no more than 10% by weight of flavonoids (optionally containing 0.05-5% by weight of flavonoids, further optionally containing 0-2% by weight of flavonoids, further optionally containing 0.05-2% by weight of flavonoids, further optionally containing 0.5-1.5% by weight of flavonoids, further optionally containing 0-1% by weight of flavonoids, further optionally containing 0.05-1% by weight of flavonoids), And / or contains amino acids at a weight content not exceeding 50% (optionally containing 0-30% amino acids, further optionally containing 0-25% amino acids, further optionally containing 0-20% amino acids, further optionally containing 0-5% amino acids, further optionally containing 3-25% amino acids, or further optionally containing 5-20% amino acids). And / or other components (optionally 0-25% by weight, further optional 0-20% by weight, further optional 0-15% by weight, further optional 0-11% by weight, further optional 2-20% by weight, further optional 4-8% by weight).

2. The use of the mulberry extract according to claim 1 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that, The treatment and / or prevention of diabetic vascular complications is manifested in improving vascular endothelial cell function impairment; preferably, it is manifested in improving any one or more of the following: (1) Promotes the proliferation of vascular endothelial cells; (2) Promotes the migration of vascular endothelial cells; (3) Promotes angiogenesis; (4) Inhibits apoptosis of vascular endothelial cells; (5) Promotes NO accumulation in endothelial cells; (6) Upregulate the expression of vasoactive factors, preferably including CD31 (platelet-endothelial cell adhesion factor-1), VEGF (vascular endothelial growth factor) (preferably VEGF-A) and / or eNOS (endothelial nitric oxide synthase); (7) Relieve vascular endothelial inflammation, preferably by inhibiting the adhesion of vascular endothelial cells to monocytes, and more preferably by inhibiting the secretion of vascular endothelial cell adhesion factors, said adhesion factors including P-selectin and / or E-selectin; (8) Increase the number of mitochondria in vascular endothelial cells.

3. The use of the mulberry extract according to claim 2 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that, The improvement in vascular endothelial cell function is also manifested in promoting the expression of NRF2 (nuclear factor erythroid lineage 2-related factor 2) and / or HO-1 (heme oxygenase 1).

4. The use of the mulberry extract according to claim 1 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that, The vascular complications of diabetes include diabetic foot or diabetic atherosclerosis.

5. The use of the mulberry extract according to claim 4 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that, The treatment and / or prevention of diabetic foot is manifested in any one or more of the following: (1) Promotes wound healing, including promoting wound regeneration, re-epithelialization, and / or collagen production. (2) Reduces cell apoptosis in the wound area (3) Promotes cell migration and / or proliferation in the wound area. (4) Promotes angiogenesis, remodeling and / or repair in the wound area, preferably promotes the expression of CD31, VEGF (preferably VEGF-A) and / or eNOS.

6. The use of the mulberry extract according to claim 4 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that, The treatment and / or prevention of diabetic atherosclerosis is manifested in any one or more of the following: (1) Reduce the formation of arterial plaques (2) Improves arterial blood flow, (3) Relieve vascular endothelial inflammation, preferably by reducing the level of vascular endothelial cell adhesion factors, including P-selectin and / or E-selectin.

7. The use of the mulberry extract according to any one of claims 1-6 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that, Based on the sum of the weight percentages of all components of the mulberry extract being 100%, the weight percentages of each component in the mulberry extract are as follows: Preferably, with the sum of the weight percentages of all components of the mulberry extract being 100%, the weight percentages of each component in the mulberry extract are as follows: Preferably, with the sum of the weight percentages of all components of the mulberry extract being 100%, the weight percentages of each component in the mulberry extract are as follows: Preferably, with the sum of the weight percentages of all components of the mulberry extract being 100%, the weight percentages of each component in the mulberry extract are as follows: Preferably, with the sum of the weight percentages of all components of the mulberry extract being 100%, the weight percentages of each component in the mulberry extract are as follows: Preferably, with the sum of the weight percentages of all components of the mulberry extract being 100%, the weight percentages of each component in the mulberry extract are as follows: Preferably, with the sum of the weight percentages of all components of the mulberry extract being 100%, the weight percentages of each component in the mulberry extract are as follows: More preferably, based on the sum of the weight percentages of all components of the mulberry extract as 100%, the weight percentages of each component in the mulberry extract are as follows: More preferably, based on the sum of the weight percentages of all components of the mulberry extract as 100%, the weight percentages of each component in the mulberry extract are as follows:

8. The use of the mulberry extract according to any one of claims 1-7 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that, The alkaloids include 1-deoxynojirimycin (DNJ), N-methyl-1-deoxynojirimycin, fagomine (FAG), 3-epi-fagomine, 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), calystegin B2, calystegin C1, and 2-oxo-(α-D-galactoside) One or more of the following: 2-O-(α-D-galactopyranosyl)-1-deoxynojirimycin, 6-O-(β-D-glucopyranosyl)-1-deoxynojirimycin, and 1,4-dideoxy-1,4-imino-(2-O-β-D-glucopyranosyl)-D-arabinitol; Optionally, the weight percentage of DNJ is not less than 50% of the total alkaloids (optionally 60-99%).

9. The use of the mulberry extract according to any one of claims 1-8 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that, The preparation of the mulberry extract includes the following steps: 1) Prepare a crude extract of mulberry plants; 2) Separate the crude extract with a cation exchange resin and / or optionally an anion exchange resin to obtain a resin eluent; optionally, step 3) subject the resin eluent of step 2) to alcohol precipitation and collect the supernatant; 4) Concentrate and / or dry the supernatant; optionally, concentrate and / or dry the resin eluent of step 2) before alcohol precipitation.

10. The use of the mulberry extract according to any one of claims 1-9 in the preparation of a medicament for treating and / or preventing diabetic vascular complications, characterized in that, The mulberry extract is effective in humans or mammals; Optionally, the drug is an oral dosage form; optionally, the drug is a tablet, capsule, oral solution, oral emulsion, pill, granule, syrup or powder.

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